Hyaluronic acid containing compositions for prevention of the formation of post-surgical scars and post-surgical adhesions

ABSTRACT

The present invention relates to pharmaceutical compositions enhancing the therapeutic effect of biologically active peptides, especially peptides derived from human lactoferrin. The compositions are useful for the treatment and/or prevention of wounds, scars, and post surgical adhesions.

RELATED APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 14/458,637, filed on Aug. 13, 2014, which is aDivisional Application of U.S. patent application Ser. No. 13/143,875,filed on Jul. 8, 2011. U.S. patent application Ser. No. 13/143,875 isthe 35 U.S.C. § 371 national stage filing of International ApplicationNo. PCT/EP2010/050284, filed on Jan. 12, 2010, which claims priority toSwedish Patent Application No. 0900031-6, filed on Jan. 13, 2009. Theentire teachings of U.S. Ser. Nos. 14/458,637 and 13/143,875 areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions enhancingthe therapeutic effect of biologically active peptides, especiallypeptides derived from human lactoferrin. The compositions are useful forthe treatment and/or prevention of wounds, scars, and post surgicaladhesions.

BACKGROUND

Peritoneal adhesions are fibrous tissue connections between abdominalstructures following surgical trauma or other types of injury. Generalabdominal, vascular, gynaecological, urological and orthopaedic surgerymay lead to adhesion formation in up to 95% of patients (Ellis et al.1999. Adhesion-related hospital readmissions after abdominal and pelvicsurgery: a retrospective cohort study. Lancet 353, 1476-1480).Post-surgical adhesions are considered the main cause of small bowelobstruction (Menzies et al. 2001. Small bowel obstruction due topostoperative adhesions: treatment patterns and associated costs in 110hospital admissions. Ann R Coll Surg Enql 83, 40-46.), a well-knownaetiology of secondary infertility in females (Marana et al. 1995.Correlation between the American Fertility Society classifications ofadnexal adhesions and distal tubal occlusion, salpingoscopy, andreproductive outcome in tubal surgery. Fertil Steril 64, 924-929) aswell as a possible cause of postoperative pain (Paajanen et al. 2005.Laparoscopy in chronic abdominal pain: a prospective nonrandomizedlong-term follow-up study. J Clin Gastroenterol 39, 110-114). More than30% of individuals undergoing lower abdominal surgery are readmitted fordisorders directly or possibly related to adhesion formation at someperiod of their life (Lower et al. 2000. The impact of adhesions onhospital readmissions over ten years after 8849 open gynaecologicaloperations: an assessment from the Surgical and Clinical AdhesionsResearch Study. Biog 107, 855-862.).

In many decades, attempts to reduce post-surgical adhesions by reducingsurgical trauma (avoiding desiccation, gentle tissue handling,meticulous hemostasis) and contamination of the abdominal cavity withforeign materials (using starch-free gloves, lint-free gauze andabsorbable sutures) have been done (Holmdahl et al. 1997. Adhesions:pathogenesis and prevention-panel discussion and summary. Eur J SurgSuppl, 56-62.). Importantly, the laparoscopic techniques are notsufficient to overcome the problem of post-operative adhesion formation(Duron et al. 2000. Prevalence and mechanisms of small intestinalobstruction following laparoscopic abdominal surgery: a retrospectivemulticenter study. French Association for Surgical Research. Arch Surg135, 208-212). Thus, intra-peritoneal adhesions remain a major clinicalissue and it is now believed that future improvements may onlymarginally be influenced through superior surgical technique. Instead,the focus is to develop dedicated products for prevention of adhesionformation, which are administrated in connection to the surgicalintervention.

Most of the therapeutic strategies tested in prevention of adhesions aremedical device products. Different types of physical barriers have beenevaluated, where the biodegradable films applied during the interventionare used to keep the injured abdominal surfaces separated during thecritical period of peritoneal healing. The two most widely usedadhesion-reducing barriers are Interceed (Johnson & Johnson MedicalInc., Arlington, Tex.) and Seprafilm™ (Genzyme, Cambridge, Mass., USA).Seprafilm™, composed of sodium hyaluronic acid andcarboxymethylcellulose (CMC) forms a viscous gel approximately 24-48 hafter placement, which is slowly resorbed within 1 week (Diamond, 1996.Reduction of adhesions after uterine myomectomy by Seprafilm membrane(HAL-F): a blinded, prospective, randomized, multicenter clinical study.Seprafilm Adhesion Study Group. Fertil Steril 66, 904-910; Beck, 1997.The role of Seprafilm bioresorbable membrane in adhesion prevention. EurJ Surg Suppl, 49-55). Seprafilm™ has been shown to reduce post-surgicaladhesion in clinical situation (Vrijland et al. 2002. Fewerintraperitoneal adhesions with use of hyaluronicacid-carboxymethylcellulose membrane: a randomized clinical trial. AnnSurg 235, 193-199.; Beck et al. 2003. A prospective, randomized,multicenter, controlled study of the safety of Seprafilm adhesionbarrier in abdominopelvic surgery of the intestine. Dis Colon Rectum 46,1310-1319; Tang et al. 2003. Bioresorbable adhesion barrier facilitatesearly closure of the defunctioning ileostomy after rectal excision: aprospective, randomized trial. Dis Colon Rectum 46, 1200-1207), however,the device is difficult to apply, as it adheres to gloves and organs andis brittle (DeCherney & diZerega, 1997. Clinical problem ofintraperitoneal postsurgical adhesion formation following generalsurgery and the use of adhesion prevention barriers. Surg Clin North Am77, 671-688). Additionally, Seprafilm™ increases the risk of sequelaeassociated with anastomosic leak and is not compatible with laparoscopicprocedures (diZerega et al. 2002. A randomized, controlled pilot studyof the safety and efficacy of 4% icodextrin solution in the reduction ofadhesions following laparoscopic gynaecological surgery. Hum Reprod 17,1031-1038). Interceed, composed of oxidized regenerated cellulose, istransformed into a gelatinous mass covering the injured peritoneum andhas shown efficacy in adhesion-prevention in several clinical studies(Mais et al. 1995. Prevention of de-novo adhesion formation afterlaparoscopic myomectomy: a randomized trial to evaluate theeffectiveness of an oxidized regenerated cellulose absorbable barrierHum Reprod. 10, 3133-3135; Mais et al. 1995 Reduction of adhesionreformation after laparoscopic endometriosis surgery: a randomized trialwith an oxidized regenerated cellulose absorbable barrier ObstetGynecol. 86, 512-515; Wallwiener et al. 1998. Adhesion formation of theparietal and visceral peritoneum: an explanation for the controversy onthe use of autologous and alloplastic barriers? Fertil Steril 69,132-137). However, application of Interceed requires complete hemostasisas even small amounts of intraperitoneal bleeding negates any beneficialeffect of this barrier (DeCherney & diZerega, 1997. supra). A generallimitation of using the physical barriers is the site-specificity of theproduct, requiring the surgeon to predict where adhesions will occur andwhere they would most likely cause clinical problems. As an alternativeto barriers, different fluids for intra-abdominal instillation such asicodextrin (Adept, Baxter Healthcare Corporation, IL, USA) or lactatedRingers' solution, have been administrated after the surgery in volumessufficient to allow floatation of the abdominal structures and thuspreventing the injured surfaces from reaching each other (Yaacobi et al.1991. Effect of Ringer's lactate irrigation on the formation ofpostoperative abdominal adhesions. J Invest Surg 4, 31-36; Cavallari etal. 2000. Inability of University of Wisconsin solution to reducepostoperative peritoneal adhesions in rats. Eur J Surg 166, 650-653.;diZerega et al. supra). However, the gravity causes problems bypreventing even distribution of the fluid in the abdomen. Also, thesolutions are absorbed more rapidly from the abdominal cavity than thetime required for peritoneal healing.

A limited number of pharmacologically active compounds have been testedin prevention of post-surgical adhesions. As some examples, theinflammatory component and fibroblast proliferation of the wound healingcascade has been a target of pharmacotherapy by using steroids drugs andcytotoxic drugs, respectively. However, these agents have shownambiguous efficacy and potentially serious side effects (LeGrand et al.1995. Comparative efficacy of nonsteroidal anti-inflammatory drugs andanti-thromboxane agents in a rabbit adhesion-prevention model. J InvestSurg 8, 187-194; Li et al. 2004. Synthesis and biological evaluation ofa cross-linked hyaluronan-mitomycin C hydrogel. Biomacromolecules 5,895-902).

Due to the limited efficacy and difficult handling of the testedtherapies, the vast majority of surgical interventions performed inabdominal cavity today, do not apply any products to prevent adhesionformation and the post-operational adhesions continue to cause sufferingfor the patients and present the major cost for society (Ray et al.1998. Abdominal adhesiolysis: inpatient care and expenditures in theUnited States in 1994. J Am Coll Surg 186, 1-9.; 2005).

The object of the present invention is to provide a means which has theability to prevent the formation of post-operative adhesion formationwithout having the unwanted side effects of the currently availablepharmaceutical compositions, devices and procedures.

DESCRIPTION OF THE INVENTION

The present inventors describe the novel approach to prevent formationof intra-abdominal adhesions using biologically active peptides derivedfrom human lactoferrin formulated in a pharmaceutical compositionenhancing the therapeutic effect of the peptides. The biologicallyactive peptides exhibit an inhibitory effect on the most importanthallmarks of scar formation: reducing risk for infections, prohibitinginflammation and promoting fibrinolysis. The peptides are formulatedtogether with the naturally occurring hydrophilic polymer hyaluronicacid, which provides slow release properties of the drug and contributesto the final results by physical barrier effect. Using a sidewalldefect-cecum abrasion model in rats, generally accepted as adequatenon-clinical predictor of clinical efficacy for anti-adhesive drugs, itis shown that biologically active peptides derived from humanlactoferrin formulated in hyaluronic acid significantly reducepost-surgical intra-abdominal adhesions. The improved effect of thepeptides when formulated in hyaluronic acid is unexpected, andsignificantly synergistic as compared to the effect of the peptides andthe effect of hyaluronic acid given independently.

Accordingly, the present invention relates to pharmaceuticalcompositions enhancing the therapeutic effect of biologically activepeptides, especially peptides derived from human lactoferrin.

One aspect the present invention provides a pharmaceutical compositionfor the treatment and/or prevention of wounds, scars, and post surgicaladhesions comprising i) one or more biologically active peptides derivedfrom human lactoferrin, and ii) a high molecular weight hyaluronic acid.

Another aspect of the present invention provides use of a i) one or morebiologically active peptides derived from human lactoferrin, and ii) ahigh molecular weight hyaluronic acid for the manufacture of apharmaceutical composition for the treatment and/or prevention ofwounds, scars, and post surgical adhesions.

Yet another aspect of the present invention provides a method for thetreatment, prophylaxis and/or prevention wounds, scars, and postsurgical adhesions comprising the administration of a pharmaceuticalcomposition comprising i) one or more biologically active peptidesderived from human lactoferrin, and ii) a high molecular weighthyaluronic acid, to a subject in need of such treatment.

By “a biologically active peptide derived from human lactoferrin” ismeant a biologically active peptide comprising at least one sequencemotif which in part or in full is derived from the sequence of humanlactoferrin, wherein this sequence motif can comprise one or more aminoacid substitutions.

By “biologically active” peptides is meant peptides that have one ormore activities, such as anti-inflammatory activity, immunomodulatoryactivity, fibrinolytic activity, anti-angiogenetic activity, andanti-microbial activity such as anti-bacterial activity, anti-viralactivity, or anti-fungal activity.

Biologically active peptides suitable to be used according to thepresent invention are described in e.g. PCT/EP2008/064062,PCT/EP2008/065186, WO 00/01730, the corresponding EP 1095061 and U.S.Pat. No. 7,253,143, which hereby are incorporated by reference.

The biologically active peptide can be selected from peptides comprisingthe amino acid sequence

(SEQ ID NO: 1) Phe-X1-X2-X3-X4-X5-X6-X7-Lys-Val-Argwherein amino acid X1 is Gln or Ala, amino acid X2 is Trp or Leu, aminoacid X3 is Gln, Ala, Orn, Nle or Lys, amino acid X4 is Arg, Ala or Lys,amino acid X5 is Asn, Ala, Orn or Nle, amino acid X6 is Met, Ala or Leu,amino acid X7 is Arg, Ala or Lys.

Preferably the biologically active peptide can be selected from peptidesaccording to formula (I) and peptides according to formula (II)

Formula (I) R1-Cys-Phe-X1-X2-X3-X4-X5-X6-X7-Lys-Val-Arg-R2

wherein R1 is either no amino acid, Lys or a peptide sequence selectedfromGly-Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys. . .

(SEQ ID NO:2) and N-terminally truncated fragments thereof including

(SEQ ID NO: 57) Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys, (SEQ ID NO: 58)Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln- Pro-Glu-Ala-Thr-Lys,(SEQ ID NO: 59) Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys, (SEQ ID NO: 60)Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu- Ala-Thr-Lys,(SEQ ID NO: 61) Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys, (SEQ ID NO: 62)Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr- Lys, (SEQ ID NO: 63)Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys, (SEQ ID NO: 64)Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys, (SEQ ID NO: 65)Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys, (SEQ ID NO: 66)Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys, (SEQ ID NO: 67)Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys, (SEQ ID NO: 68)Ser-Gln-Pro-Glu-Ala-Thr-Lys, (SEQ ID NO; 69) Gln-Pro-Glu-Ala-Thr-Lys,(SEQ ID NO: 70) Pro-Glu-Ala-Thr-Lys, (SEQ ID NO: 71) Glu-Ala-Thr-Lys,Ala-Thr-Lys, Thr-Lys-;

and wherein R2 is either no amino acid, Gly or a peptide sequenceselected from

(SEQ ID NO: 3) Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys-Argand C-terminally truncated fragments thereof including

(SEQ ID NO: 72) Gly-Pro-Pro-Val-Ser-Cys-Ile-Lys, (SEQ ID NO: 73)Gly-Pro-Pro-Val-Ser-Cys-Ile, (SEQ ID NO: 74) Gly-Pro-Pro-Val-Ser-Cys,(SEQ ID NO: 75) Gly-Pro-Pro-Val-Ser, (SEQ ID NO: 76) Gly-Pro-Pro-Val,Gly-Pro-Pro,  and Gly-Pro

wherein amino acid X8 is Gly, Lys, Glu or Asp;when X8 is Gly then R3 is Ser-(Arg)_(n)-X9 and the bond α is a peptidebond between the carboxyl group of Gly and the amino group of Ser;when X8 is Lys then R3 is X9-(Arg)_(n)-Ser and the bond a is an amidebond between the s-amino group in Lys and the carboxyl group in Ser; andwhen X8 is Glu or Asp then R3 is Ser-(Arg)_(n)-X9 and the bond a anamide bond between the γ-carboxyl group of Glu or the β-carboxyl groupof Asp and the amino group of Ser; amino acid X9 is either no amino acidor Gly;and n is an integer from 1 to 10, preferably an integer from 2 to 6,preferably an integer from 4 to 6, or even more preferably an integerfrom 3 to 4;and wherein R1 is either no amino acid, Cys or a peptide sequenceselected from

(SEQ ID NO: 48) y-Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cysand N-terminally truncated fragments thereof including

(SEQ ID NO: 77) Gly-Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 78)Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 79)Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 80)Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro- Glu-Ala-Thr-Lys-Cys,(SEQ ID NO: 81) Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 82)Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala- Thr-Lys-Cys,(SEQ ID NO: 83) Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 84)(Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys- Cys, (SEQ ID NO: 85)Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 86)Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 87)Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 88)Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 89)Ser-Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 90)Gln-Pro-Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 91) Pro-Glu-Ala-Thr-Lys-Cys,(SEQ ID NO: 92) Glu-Ala-Thr-Lys-Cys, (SEQ ID NO: 93) Ala-Thr-Lys-Cys,Thr-Lys-Cys, and Lys-Cysand wherein R2 is either no amino acid, Pro or a peptide sequenceselected from

(SEQ ID NO: 49) Pro-Pro-Val-Ser-Cys-Ile-Lys-and C-terminally truncated fragments thereof including

(SEQ ID NO: 94) Pro-Pro-Val-Ser-Cys-Ile-Lys, (SEQ ID NO: 95)Pro-Pro-Val-Ser-Cys-Ile, (SEQ ID NO: 96) Pro-Pro-Val-Ser-Cys,(SEQ ID NO: 97) Pro-Pro-Val-Ser , Pro-Pro-Val,  and Pro-Pro;Even more preferably the biologically active peptide can be selectedfrom the peptides

Most preferably the biologically active peptide is selected from thepeptides

(SEQ ID NO: 4) Ac-Glu-Ala-Thr-Lys-Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys-Ile- Lys-Arg-NH₂ and(SEQ ID NO: 50) Ac-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Ser-Arg-Arg-Arg-Arg-Gly-NH₂;

Peptides comprising two cysteine residues can be in the form of a cyclicpeptide structure where the two cysteines form a cysteine bridge.

Accordingly, one preferred biologically active peptide is the peptide

When present, it may be advantageous to replace the amino acid Cys by anacetamidomethyl-cysteine (indicated as CysM) in order to avoid that thepeptide forms a disulphide bridge with another peptide comprising acysteine.

According to one preferred aspect of the invention the carboxy terminalend of the peptide has been capped, i.e. the free COOH at the carboxyterminal end has been transformed, e.g. by amidation into CONH₂.(indicated as —NH₂)

According to another preferred aspect of the invention the aminoterminal end of the peptide has been capped, i.e. the free NH₂ group atthe amino terminal has been transformed, e.g. by acetylation into theamide CH₃CONH— (indicated as Ac—).

According to yet another preferred aspect of the invention both thecarboxy-terminal and the amino-terminal ends of the peptide have beencapped.

In the case where a peptide according to the invention is described asbeing capped at the carboxy terminal end and/or amino terminal end, itis also possible according to the invention to use the correspondinguncapped peptide.

In the case where a peptide according to the invention is described asbeing uncapped at the carboxy terminal end and/or amino terminal end, itis also possible according to the invention to use the correspondingcapped peptide.

The advantage of the capped versions is that N- and C-terminal aminoacids of these peptides are neutral and uncharged and thus has changedelectrostatic properties. Assuming that the receptors bind thecorresponding sequences of human lactoferrin where there are no N- and Cterminal charges, the capped peptides should bind better as they in thisrespect resemble the native protein more than uncapped peptides.

Preferably the biologically active peptide is present in thepharmaceutical composition at a concentration between 0.1 mg/ml and 100mg/ml, most preferably between 0.5 mg/ml and 25 mg/ml.

The biologically active peptide can be present in the form of apharmaceutical acceptable salt.

Preferably the high molecular weight hyaluronic acid has a molecularweight higher than 300,000 Da, most preferably higher than 800,000 Da.

Preferably the high molecular weight hyaluronic acid is present in thepharmaceutical composition at a concentration between 0.1 and 10% (w/w),most preferably between 0.5 and 2.5% (w/w).

The high molecular weight hyaluronic acid can be present in the form ofa pharmaceutical acceptable salt.

The pharmaceutical compositions according to the invention can be usedto prevent the formation of post surgical scars, adhesions, keloids inconnection with surgical procedures on various tissues such as skin,muscles, tendons, nervous tissue, blood vessels, and at differentlocations of the body such as eyes, ears, vocal cord, hand, spinal cord,intra-abdominal cavity, intra-thoracic cavity, intra-cranial cavity,oral cavity, gynaecological procedures, endometrios, phimosis.

The present inventors have unexpectedly found that the biological effectof the peptides derived from human lactoferrin can be significantlyenhanced if the peptides are administered in a pharmaceuticalcomposition comprising the peptide together with a high molecular weighthyaluronic acid.

This enhancement can not be explained only by a possible effect of thehyaluronic acid as such, but is due to an unexpected synergistic effect.

DESCRIPTION OF FIGURES

FIG. 1. The behaviours of PXL01 loaded sodium hyaluronate gels at 37° C.

The behaviours of PXL01 loaded sodium hyaluronate gels at 37° C. Theconcentration of PXL01 is 6 mg/ml in 1.5% sodium hyaluronate solution.The cumulative drug released was expressed as the % drug released attime t. The data are shown as mean±SDV of three independent productpreparations with the moving average trendline added.

FIG. 2. PXL01 prevents adhesion formation in rat model of abdominalsurgery.

(A) The incidence of adhesion formation between the injury sites ofabdominal wall and cecum, presented as a percentage of animalsdeveloping wall to wall adhesion connecting these injuries in eachgroup. (B) The cumulative scoring scale showing the total number ofadhesions found in the abdominal cavity presented as mean±SEM. (C) Theadhesion scores according to the Nair scale presented as mean±SEM(scoring criteria listed in Examples). (D) Percentage of animals withoutany adhesion formation in the abdominal cavity in each group. (E) Weightchange during the 6 survival days after surgery presented as percentageof initial weight. n(control)=20, n(1 administration of 0.5 ml PXL01 (6mg/ml) in dH2O)=10, n(3 administrations of 0.5 ml PXL01 (2 mg/ml) indH2O in connection to the operation and 24 and 48 h post-surgery)=18,n(1 administration of 1.5% sodium hyaluronate)=20, n(1 administration of1 ml PXL01 (1.5 mg/ml) in 1.5% sodium hyaluronate)=10, n(1administration of 1.5 ml PXL01 (6 mg/ml) in 1.5% sodiumhyaluronate)=10). Statistical significance was estimated by Fisher'sexact test (A, D) or by non-parametric Mann Whitney test (B, C). *,p<0.05; **, p<0.01 indicate statistical difference compared to thesurgical control group of animals. Adm, administration; SH, sodiumhyaluronate, dH20, distilled water.

EXAMPLES Experimental

Peptide

The peptide PXL01 (SEQ ID NO:56) was used in the experiments.

Preparation of PXL01 in Sodium Hyaluronate Hydrogels

PXL01 dissolved in sodium chloride solution was added to 2.5% sodiumhyaluronate solution at a volume ratio of 2/5 PXL01 solution and 3/5sodium hyaluronate solution, to obtain 1.5 or 6 mg/ml PXL01 in 1.5%sodium hyaluronate. The solutions were homogenized by drawing themixtures several times through 2.1 mm diameter needles.

Characterization of Formulated Product

PXL01 concentration and homogeneity in sodium hyaluronate weredetermined by high performance liquid chromatography with UV detector(Agilent model 1100) at 220 nm. The analytical column used was a Vydac218TP (C18, 5 μm, 250×4.6 mm). The mobile phases used (0.1% TFA in watercontaining 1% acetonitrile (solvent A) and 0.1% TFA in acetonitrile(solvent B)) were run at a gradient with a flow rate of 1.0 ml/min.Diluted PXL01 standards were applied to create calibration curves.

Samples were prepared by adding hyaluronidase solution (Hyaluronidasefrom Streptomyces hyalurolyticus, Sigma-Aldrich, St Louis, Mo.) with anenzyme activity of 500 units/ml to sample solutions. The mixtures wereagitated for 2 h at room temperature and samples were diluted as neededwith TFA in water, followed by additional mixing. The samples werecentrifuged at 7000 rpm for 5 min before injection to the column.

In Vitro Release System Setup

0.25 ml of the formulated product was placed into the well of the tissueculture plate (24-Flat Well Tissue Culture Plate, Techno PlasticProducts AG), resulting in a thin film of approximately 1.3 mm. Theplates were placed into thermostat (37° C.) for 1 h to allow the productto reach the temperature of 37° C. 0.5 ml of the release medium (PBS, pH7.4) re-equilibrated at 37° C. was carefully layered over the surface ofthe gel and the tissue culture plates were transferred into athermostatic shaker (60 rpm, 37° C.). At predetermined time intervals,10 microl aliquots of the aqueous solution were withdrawn from therelease media. The concentration of PXL01 released was monitored atwavelength of 230 nm using a spectrophotometric measurement. Because themeasurement of absorbance at 230 nm could detect the peptide as well asdissolved sodium hyaluronate in the release medium, a control releasemedium was used which has the same amount of sodium hyaluronate withoutany PXL01 as that of sodium hyaluronate with the drug.

Animal Models for Assessment of Post-Surgical Adhesion Prevention

Female Sprague-Dawley rats (200-250 g, Charles River Laboratories,Sulzfeldt, Germany) were kept in a 12 hours light-dark cycle and werecared for in accordance with regulations for the protection oflaboratory animals. The study was performed after prior approval fromthe local ethical committee.

Cecum abrasion and excision of the abdominal wall were performed toinduce de novo adhesions as described previously (Harris et al. 1995.Analysis of the kinetics of peritoneal adhesion formation in the rat andevaluation of potential antiadhesive agents. Surgery 117, 663-669).Briefly, the rats were anaesthetized with isoflurane (Isoba®vet,Shering-Plough Animal Health, Farum, Denmark) and buprenorfin (48microg/kg, Temgesic, Shering-Plough, Brussels, Belgium) was given aspost-operative pain reliever. A 5-cm-long midline incision of theabdomen was performed and a rectangle full thickness injury (5 mm×25 mm)was made on the peritoneal wall through both the parietal peritoneum andthe muscular fascia. Also, an area of the serous membrane on the bothsides of the cecum, approximately 10 mm×15 mm, was gently rubbed usingcotton gauze until petechial hemorrhages appeared. The rats wererandomized to untreated control group or treated groups. Excessive bloodfrom the injury was removed and the test substance was applied over theabraded areas using a syringe. The laparotomy wound was closed with acontinuous suture and the skin was closed with metal clips (ApposeULC35W, TycoHealthcare Group LP, Norwalk, Conn., US). The animals werekilled 6 days after surgery with an overdose of pentobarbital sodium(Pentobarbital vet, APL, Stockholm, Sweden). The abdomen was opened andthe adhesions were scored by an evaluator blinded to the treatment. Theincidence of adhesions between abdominal incision and the abraded cecumwas quantified as a percentage of animals developing wall to walladhesions connecting these injuries, in each group. Additionally, tocomprehensively evaluate the total number of adhesions formed in theabdominal cavity, including the adhesions remote from the surgicaltrauma, two different grading schemes were used. The cumulative scoringscale described by Bothin (Bothin et al. 2001. The intestinal florainfluences adhesion formation around surgical anastomoses. Br J Surg 88,143-145) assigns the total number of adhesions present in the abdominalcavity: one point is given to each adhesion observed and the points areadded to form the score. The adhesion scoring scale according to Nair(Nair et al. 1974. Role of proteolytic enzyme in the prevention ofpostoperative intraperitoneal adhesions. Arch Surg 108, 849-85)incorporates both the total number of adhesions and the incidence ofadhesions between target organs, while a higher grading is given to thelatter one (0, no adhesions; 1, single band of adhesions from theviscera to the target organ; 2, two bands of adhesions from the viscerato the target organ; 3, more than two adhesive bands from the viscera tothe target organ, 4, viscera directly adherent to abdominal wall,irrespective of number and extent of adhesive bands). Finally, thepercentage of rats free from any abdominal adhesions was assessed ineach group. Any possible signs of peritoneal inflammation (erythemaand/or edema) or disrupted wound healing were recorded in connection tothe necropsies. As a general marker for well being, the body weights ofanimals before and 6 days after the surgery were compared.

Large Bowel Anastomosis Model in the Rat

Female Sprague Dawley rats (200-250 g, Charles River Laboratories,Sulzfeldt, Germany) were kept at a 12 hours light-dark cycle and werecared for in accordance with regulations for the protection oflaboratory animals. The study was conducted after prior approval fromthe local ethical committee. Anaesthesia was induced with isoflurane(Isoba®vet, Shering-Plough Animal Health, Farum, Denmark) and the ratsreceived buprenorfin (48 microg/kg; Temgesic, Shering-Plough, Brussels)intramuscularly for post-operative pain relieve and Bimotrim (80 mg/kg;Bimeda, UK,) subcutaneously before the surgery.

The abdominal wall was shaved and a midline laparatomy of approximately3 cm was performed. The colon was exposed and transected 2 cm distal ofcecum. A seromuscular end-to-end anastomosis was performed with 8interrupted sutures using 6/0 monocryl (Y432H, Ethicon Inc, St-StevensWoluwe, Belgium) thread. A macaroon was placed in colon at anastomosisas stent during suturing. The rats were randomly divided into groupsreceiving PXL01 (6 mg/ml) in 1.5% sodium hyaluronate covering theanastomosis and surrounding peritoneum (n=8) or no treatment (n=8). Theabdomen was closed with a continuous suture (4-0 monocryl, Y3100H,Ethicon Inc.) in the muscular layer and with staplers in the skin. 2 mlisotonic saline was administered subcutaneously to prevent dehydration.

The animals received additional doses of buprenorfin (24 microg/kg;Temgesic, Shering-Plough, Brussels) subcutaneously two times per day fortwo days after surgery. The animals were killed 7 days after surgerywith an overdose of pentobarbital sodium (Pentobarbital vet, APL,Stockholm, Sweden). The abdomen was opened and a 4-cm-long intestinalsegment was resected with the anastomosis area located in the middle. Atube connected to a pressure monitor was inserted into one side of theintestinal segment and the other side was ligated at the end. Theintestinal segment was placed immediately under isotonic sodiumchloride, stained saline was infused through the tube into theintestinal segment, and the intraluminar pressure was monitored using aGrass recorder (Grass Instruments Co, Quincy, Ohio, USA). The maximumpressure prior to anastomotic burst was recorded as the burst pressure.The appearance of stained saline around the anastomosis indicated thetime point for the burst. The evaluator was blinded to the treatmenteach animal received.

Results

PXL01 Release Behaviour in Sodium Hyaluronate

PXL01 dissolved in sodium chloride solution was mixed with the sodiumhyaluronate solution resulting in homogenous PXL01-containing hydrogel.The in vitro release experiments revealed a burst release of PXL01 fromthe sodium hyaluronate gel formulation with approximately 70% of PXL01released within 1 hour (FIG. 1). Release behaviour characterized by aninitial burst is already demonstrated for other soluble compoundsformulated in sodium hyaluronate (Sherwood et al. 1992. Controlledantibody delivery systems. Biotechnology (N Y) 10, 1446-1449). This mayhave a functional use in providing an initial dose during drug delivery,minimizing any lag period. Importantly, the release profiles of PXL01from the formulated products prepared in three independent occasionswere largely overlapping indicating that preparation of PXL01-loadedsodium hyaluronate gels is highly reproducible (FIG. 1).

Prevention of Peritoneal Adhesions by PXL01

The sidewall defect-cecum abrasion model in rat (Arnold et al. supra)was used to elucidate the anti-adhesion effect of PXL01. This modelproduces reliable and consistent adhesions between the two injuredsurfaces if no treatment is given, with 85% of the rats in the controlgroup developing direct cecum-peritoneal wall adhesions (FIG. 2A). Nosignificant reduction in adhesion formation was observed when 3 mg ofPXL01 in water solution was administrated as a single dose in connectionto the surgery (FIGS. 2A-D). However, animals treated with 3 doses of 1mg of PXL01 in water solution demonstrated marked reduction in adhesionformation compared with the control group of rats (FIGS. 2A, C). Theseresults indicate that slow release of PXL01 in the surgical area isbeneficial, compared to the single treatment with the water solution ofthe peptide.

Sodium hyaluronate was chosen as carrier to achieve controlled releaseof PXL01. PXL01 appears readily soluble and sufficiently stable insodium hyaluronate, also the PXL01-containing sodium hyaluronatehydrogel is bioadhesive and easy to apply to the surgical area using asyringe. When PXL01 was applied in 1.5% high molecular weight sodiumhyaluronate formulation, the formation of abdominal adhesions wassignificantly reduced, compared with the control group. There was a4-fold reduction according to the cumulative adhesion scoring scale(FIG. 2B) and more than 3-fold reduction of the adhesion score accordingto Nair (FIG. 2C). 60% of animals treated with 6 mg/ml PXL01 in sodiumhyaluronate were completely free from adhesions compared with 5% of theanimals in control group and 20% of animals in the group treated withsodium hyaluronate (FIG. 2D). By several scoring scales, sodiumhyaluronate per se was shown to reduce adhesion formation, presumablydue to the physical barrier effect (Burns et al. 1995. Prevention oftissue injury and postsurgical adhesions by precoating tissues withhyaluronic acid solutions. J Surg Res 59, 644-652).

No treatment-related adverse effects were recorded during the studyregarding the wound healing or peritoneal inflammation assessed duringnecropsies. Also, the average body weight of the rats in the treatmentgroups was increased compared to their pre-surgical weights, althoughthe difference compared to the control group did not reach statisticalsignificance (FIG. 2E). Importantly, PXL01 in sodium hyaluronateadministered around the intestinal anastomosis did not reduce thehealing potential as estimated by the burst pressure of anastomosismeasured 7 days after the surgery (burst pressure for the treatmentgroup (n=8) 206.3±14.3 mm Hg versus 197.4±9.6 mm Hg in the sham group(n=8)).

The ability of PXL01 to prevent adhesions was limited in water solution(FIGS. 2A-D), possibly due to the fact that the peptide is rapidlyeliminated from the peritoneum. However, the peptide was highlyeffective formulated in sodium hyaluronate (FIGS. 2A-D), causingsignificant reduction of adhesions according to different grading scalesencompassing both the adhesions formed between the two injured surfacesas well as in the abdominal areas remote form the site of application.Sodium hyaluronate, a natural component of extracellular matrix, iscatabolized locally or carried to lymph notes or the generalcirculation, from where it is cleared by the endothelial cells of theliver (Fraser et al. 1988. Uptake and degradation of hyaluronan inlymphatic tissue. Biochem J 256, 153-158; Laurent & Fraser 1992.Hyaluronan. Faseb J 6, 2397-2404). Sodium hyaluronate is likely toenhance the effect of PXL01 by maintaining local concentrations of thedrug through controlled release. In vitro experiments indicate arelatively brief period of PXL01 release from sodium hyaluronate(FIG. 1) suggesting that the duration of the drug release required foradhesion prevention in vivo may be rather limited. This is in line withthe previous evidence that the critical events in adhesion formation inabdominal cavity occur in the first 36 h (Harris et al. 1995. Analysisof the kinetics of peritoneal adhesion formation in the rat andevaluation of potential antiadhesive agents. Surgery 117, 663-669).Previously, several carrier systems based on microparticles have beenshown to induce adhesions or cause inflammation (Hockel et al. 1987.Prevention of peritoneal adhesions in the rat with sustainedintraperitoneal dexamethasone delivered by a novel therapeutic system.Ann Chir Gynaecol 76, 306-313; Kohane et al. 2006. Biodegradablepolymeric microspheres and nanospheres for drug delivery in theperitoneum. J Biomed Mater Res A 77, 351-361). No obvious adverse eventssuch as listlessness, peritoneal inflammation or inhibition of woundhealing were observed in animals treated with PXL01 at anyconcentration. At the time of sacrifice all treatment groups hadmaintained or exceeded their pre-surgery weights (FIG. 2E). Importantly,PXL01 in sodium hyaluronate administered around the intestinalanastomosis did not interfere with the healing potential of theanastomosis.

In summary, the present inventors describe an unexpected observationthat the biological effect of the lactoferrin-derived peptides onprevention of post-surgical adhesion formation can be significantlyenhanced if the peptides are administered in a pharmaceuticalcomposition comprising the peptide together with a high molecular weighthyaluronic acid. The effect is significantly synergistic as compared tothe effect of the peptide and the effect of hyaluronic acid givenindependently. Previously, several carrier systems based onmicroparticles have been shown to induce adhesions or cause inflammation(Hockel et al. 1987. supra; Kohane et al. 2006. supra). Also,applications of physical barriers for adhesion prevention have beenshown to lead to adverse effects such as anastomosis leak, due tointerference with the wound healing process (diZerega et al. 2002.supra). In the present study, sodium hyaluronate was shown not toincrease adhesions but rather to act synergistically to lactoferrinpeptides in adhesion prevention. Importantly, administration of thepeptides in sodium hyaluronate was not associated with any safetyconcern regarding healing of anastomosis and thus, the productdemonstrated the superior safety profile compared to the previouslydescribed anti-adhesive agents. The peptide-loaded sodium hyaluronategel is easy to handle and administrate and is compatible with laparatomyor laparoscopy. Taken together, the product is expected to givecomprehensive adhesion prevention regime preventing not only theadhesions which form at sites of operative procedures, but also de novoadhesions that form to sites not directly involved in surgery due tounintentional tissue injury during surgical manipulation, withoutcausing any adverse effects on healing.

The invention claimed is:
 1. A pharmaceutical composition comprising inadmixture: (i) the peptide PXL01 (SEQ ID NO:56), or a pharmaceuticallyacceptable salt thereof; and (ii) a high molecular weight hyaluronicacid having an average molecular weight higher than 300 000 Da, or apharmaceutically acceptable salt thereof.
 2. The pharmaceuticalcomposition according to claim 1, wherein the high molecular weighthyaluronic acid has an average molecular weight higher than 800 000 Da.3. The pharmaceutical composition according to claim 1, wherein thepeptide PXL01 (SEQ ID NO:56) is present at a concentration of between0.1 mg/ml and 100 mg/ml.
 4. The pharmaceutical composition according toclaim 2, wherein the peptide PXL01 (SEQ ID NO:56) is present at aconcentration of between 0.1 mg/ml and 100 mg/ml.
 5. The pharmaceuticalcomposition according to claim 3, wherein the peptide PXL01 (SEQ IDNO:56) is present at a concentration of between 0.5 mg/ml and 25 mg/ml.6. The pharmaceutical composition according to claim 4, wherein thepeptide PXL01 (SEQ ID NO:56) is present at a concentration of between0.5 mg/ml and 25 mg/ml.
 7. The pharmaceutical composition according toany one of claims 1-6, wherein the high molecular weight hyaluronic acidis present at a concentration of between 0.1 and 10% (w/w).
 8. Thepharmaceutical composition according to claim 7, wherein the highmolecular weight hyaluronic acid is present at a concentration ofbetween 0.5 and 2.5% (w/w).